The Hedgehog (Hh) signaling pathway is integral to tissue patterning during fetal development and oncogenesis in children and adults. Genetic screens have revealed several of the signaling proteins that regulate Hh target gene expression, which in mammals include the Patched family of Hh receptors (Ptc1 and Ptc2), the transmembrane protein Smoothened (Smo), the cytoplasmic negative regulator Suppressor of Fused (Su(fu)), and the Gli family of transcription factors (Gli1, Gli2, and Gli3). These genes are potential targets for next-generation chemotherapies, and compounds that inhibit Smo have demonstrated efficacy in mouse models of Hh pathway-dependent tumors, including basal cell carcinoma, medulloblastoma, pancreatic adenocarcinoma, and prostate cancer. It has become increasingly apparent, however, that oncogenic dysregulation of the Hh pathway can often involve genetic and/or epigenetic perturbations downstream of Smo, and other signaling pathways appear to promote Hh target gene expression in tumors in a Smo-independent manner. Such cancers will not be responsive to Smo inhibitors, yet to date, essentially all known Hh pathway antagonists target this transmembrane protein. This application describes mechanistic and in vivo studies of a new Hh pathway inhibitor called gantamine (Gli antagonist amine), which was discovered by a high-throughput screen of over 120,000 compounds. Gantamine is one of 14 antagonists of Gli function identified in this study and is among the most promising with respect to activity, specificity, and potency. It also appears to be mechanistically distinct from the three Hh pathway inhibitors previously reported to act downstream of Smo. Gantamine and the other Gli antagonists therefore constitute valuable tools for dissecting Hh signal transduction mechanisms and developing new chemotherapies for Hh pathway-related cancers. In particular, how Smo activity regulates Gli function remains enigmatic, and determining the mechanisms of these compounds will provide insights into this process and strategies for its pharmacological control. To achieve these goals, the structure-activity relationships for gantamine will be established by the chemical synthesis and biological evaluation of various derivatives, enabling pharmacophore optimization and probe design for target identification efforts. How gantamine and its derivatives interact with the Gli proteins, "upstream" Hh signaling proteins, and other cellular pathways known to regulate Hh target gene expression will be investigated, and various strategies for discovering the cellular target of gantamine will be pursued. Finally, the efficacy of this Gli antagonist in mouse models of normal and oncogenic Hh pathway activation will be evaluated, focusing on endochondral bone formation and the progression of medulloblastomas induced by loss of Ptc1 or Su(fu) function. PUBLIC HEALTH RELEVANCE:Dysregulation of the Hedgehog signaling pathway contributes to the formation and progression of several cancers, and chemicals that inhibit this process have potential as targeted chemotherapies. The proposed research investigates a novel Hedgehog pathway antagonist that is mechanistically distinct from other known inhibitors. These studies will determine how this compound blocks the Hedgehog pathway and evaluate its efficacy in mouse models of medulloblastoma, the most common pediatric brain tumor.